Force scanning probe microscope

ABSTRACT

A force scanning probe microscope (FSPM) and associated method of making force measurements on a sample includes a piezoelectric scanner having a surface that supports the sample so as to move the sample in three orthogonal directions. The FSPM also includes a displacement sensor that measures movement of the sample in a direction orthogonal to the surface and generates a corresponding position signal so as to provide closed loop position feedback. In addition, a probe is fixed relative to the piezoelectric scanner, while a deflection detection apparatus is employed to sense a deflection of the probe. The FSPM also includes a controller that generates a scanner drive signal based on the position signal, and is adapted to operate according to a user-defined input that can change a force curve measurement parameter during data acquisition.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention is directed to probe microscopes, and moreparticularly, a probe microscope (PM) apparatus and method for sensingtip-sample interaction forces.

[0003] 2. Description of Related Art

[0004] Developments in nanotechnology have enabled mechanicalexperiments on a broad range of samples including single molecules, suchthat fundamental molecular interactions can be studied directly. Themechanical properties of biological molecules, in particular, such asactin filaments and DNA has lead to the development of a range ofinstrumentation for conducting these studies. In this regard, systemsand methods differing in force and dynamic ranges currently being usedinclude magnetic beads, optical tweezers, glass microneedles,biomembrane force probes (BFP), scanning probe microscopy (SPM), andatomic force microscopy (AFM).

[0005] With a force sensitivity on the order of a few pico-Newtons(pN=10⁻¹²N), an AFM is an excellent tool for probing fundamental forceinteractions between surfaces. AFM has been used to probe the nature ofattractive van der Waals and attractive/repulsive electrostatic forcesbetween systems such as metal probes and insulating mica surfaces, andinsulating probes on insulating and conducting samples with materialssuch as silicon nitride, diamond, alumina, mica, glass and graphite.Other applications include the study of adhesion, friction, and wear,including the formation or suppression of capillary condensation onhydrophilic silicon, amorphous carbon and lubricated SiO₂ surfaces.

[0006] More particularly, for biological molecules, force is often animportant functional and structural parameter. Biological processes suchas DNA replication, protein synthesis, drug interaction, to name a few,are largely governed by intermolecular forces. However, these forces areextremely small. With its sensitivity in the pico-Newton scale, the SPMhas been employed to analyze these interactions. In this regard, SPMstypically are used to generate force curves that provide particularlyuseful information for analyzing very small samples.

[0007] The knowledge regarding the relation between structure, functionand force is evolving and therefore single molecule force spectroscopy,particularly using SPM, has become a versatile analytical tool forstructural and functional investigation of single bio-molecules in theirnative environments. For example, force spectroscopy by SPM has beenused to measure the binding forces of different receptor-ligand systems,observe reversible unfolding of protein domains, and investigatepolysaccharide elasticity at the level of inter-atomic bond flips.Moreover, molecular motors and their function, DNA mechanics and theoperation of DNA-binding agents such as proteins in drugs have also beenobserved. Further, the SPM is capable of making nano-mechanicalmeasurements (such as elasticity) on biological specimens, thusproviding data relative to subjects such as cellular and proteindynamics.

[0008] Another main application of making AFM force measurements is inmaterials science where the study of mechanical properties of nano-scalethin films and clusters is of interest. For example, as microstructuressuch as integrated circuits continue to shrink, exploring the mechanicalbehavior of thin films from known properties of the materials becomesincreasingly inaccurate. Therefore, continuing demand for fastercomputers and larger capacity memory and storage devices placesincreasing importance on understanding nano-scale mechanics of metalsand other commonly used materials.

[0009] PMs, including instruments such as the atomic force microscope(AFM), are devices that typically use a sharp tip and low forces tocharacterize the surface of a sample down to atomic dimensions.Generally, AFMs include a probe having a tip that is introduced to asurface of a sample to detect changes in the characteristics of thesample. In this case, relative scanning movement between the tip and thesample is provided so that surface characteristic data can be acquiredover a particular region of the sample, and a corresponding map of thesample surface can be generated. However, PMs also include devices suchas molecular force probes (MFPs) that similarly use a probe tocharacterize sample properties but do not scan.

[0010] In one application of AFM, either the sample or the probe istranslated up and down relatively perpendicularly to the surface of thesample in response to a signal related to the motion of the cantileverof the probe as it is scanned across the surface to maintain aparticular imaging parameter (for example, to maintain a set-pointoscillation amplitude). In this way, the feedback data associated withthis vertical motion can be stored and then used to construct an imageof the sample surface corresponding to the sample characteristic beingmeasured, e.g., surface topography. Other types of images are generateddirectly from the detection of the cantilever motion or a modifiedversion of that signal (i.e., deflection, amplitude, phase, friction,etc.), and are thus not strictly topographical images.

[0011] In addition to surface characteristic imaging such astopographical imaging, the AFM can probe nano-mechanical and otherfundamental properties of samples and their surfaces. Again, AFMapplications extend into applications ranging from measuring colloidalforces to monitoring enzymatic activity in individual proteins toanalyzing DNA mechanics.

[0012] When measuring biological samples, it is useful to measure, forexample, the stiffness of the sample; in one example, to separate saltcrystals from DNA or to separate the DNA from a hard surface. In U.S.Pat. No. 5,224,376, assigned to the assignee of the present invention,an atomic force microscope is described in which the system can map boththe local the stiffness (force spectroscopy) and the topography of asample. In the preferred implementation, a stiffness map of the sampleis obtained by modulating the force between the tip and sample during ascan by modulating the vertical position of the sample while keeping theaverage force between the tip and the sample constant. The bending ofthe cantilever, which is a measure of the force on the tip, is measuredby an optical detector that senses the deflection of a light beamreflected from the back of the cantilever. In a simple example, the AFMand force spectroscopy apparatus of this patent has been used to studyDNA laying on a glass surface. Modulating the force and then imaging thestiffness of the sample has the advantage that a surface such as glass,which has a rough topographic image, will have a flat stiffness image,permitting soft molecules on it such as DNA to be readily imaged.

[0013] Notably, a key element of the probe microscope is its microscopicsensor, i.e., the probe. The probe includes a microcantilever, thedesign and fabrication of which is well-known in the field, which istypically formed out of silicon, silicon nitride, or glass, and hastypical dimensions in the range of 10-1000 microns in length and 0.1-10microns in thickness. The probe may also include a “tip,” which,particularly in AFM, is typically a sharp projection near the free endof the cantilever extending toward the sample. In the more general fieldof probe microscopy, the tip may be absent or of some other shape andsize in order to control the particular type, magnitude, or geometry ofthe tip-sample interaction or to provide greater access to chemicallymodify the tip surface.

[0014] The second key element of a probe microscope is a scanningmechanism (“the scanner”), which produces relative motion between theprobe and the sample. It is well-known by those in the field that suchscanners may move either the tip relative to the sample, the samplerelative to the tip, or some combination of both. Moreover, probemicroscopes include both scanning probe microscopes in which the scannertypically produces motion in three substantially orthogonal directions,and instruments with scanners that produce motion in fewer than threesubstantially orthogonal directions (i.e.-MFP).

[0015] Turning to FIGS. 1A-1E and 2, force spectroscopy using SPM isillustrated. More particularly, FIGS. 1A-1E show how the forces betweena tip 14 of a probe 10 and a sample 16, at a selected point (X,Y) on thesample, deflect a cantilever 12 of probe 10 as the tip-sample separationis modulated in a direction generally orthogonal to the sample surface.FIG. 2 shows the magnitude of the forces as a function of sampleposition, i.e., a force curve or profile.

[0016] In FIG. 1A, probe 10 and sample 16 are not touching as theseparation between the two is narrowed by moving the sample generallyorthogonally toward the sample surface. Zero force is measured at thispoint of the tip-sample approach, reflected by the flat portion “A” ofthe curve in FIG. 2. Next, probe 10 may experience a long rangeattractive (or repulsive force) and it will deflect downwardly (orupwardly) before making contact with the surface. This effect is shownin FIG. 1B. More particularly, as the tip-sample separation is narrowed,tip 14 may “jump” into contact with the sample 16 if it encounterssufficient attractive force from the sample. In that case, thecorresponding bending of cantilever 12 appears on the force profile, asshown in FIG. 2 at the curve portion marked “B.”

[0017] Turning next to FIG. 1C, once tip 14 is in contact with sample16, the cantilever will return to its zero (undeflected) position andmove upwardly as the sample is translated further towards probe 10. Ifcantilever 12 of probe 10 is sufficiently stiff, the probe tip 14 mayindent into the surface of the sample. Notably, in this case, the slopeor shape of the “contact portion” of the force curve can provideinformation about the elasticity of the sample surface. Portion “C” ofthe curve of FIG. 2 illustrates this contact portion.

[0018] In FIG. 1D, after loading cantilever 12 of probe 10 to a desiredforce value, the displacement of the sample 16 is reversed. As probe 10is withdrawn from sample 16, tip 14 may either directly adhere to thesurface 16 or a linkage may be made between tip 14 and sample 16, suchas via a molecule where opposite ends are attached to the tip 14 andsurface 16. This adhesion or linkage results in cantilever 14 deflectingdownwards in response to the force. The force curve in FIG. 2illustrates this downward bending of cantilever 14 at portion “D.”Finally, at the portion marked “E” in FIG. 2, the adhesion or linkage isbroken and probe 10 releases from sample 16, as shown in FIG. 1E.Particularly useful information is contained in this portion of theforce curve measurement, which contains a measure of the force requiredto break the bond or stretch the linked molecule.

[0019] An example of a sample force measurement as described above isshown in FIG. 3 where two complimentary strands of DNA 20 areimmobilized on the tip and sample surfaces 22 and 24, respectively. Bymodulating the tip-sample separation, a force curve such as that shownin FIG. 2 can be generated. As a result, a quantitative measurement ofthe forces and energetics required to stretch and un-bind the DNAduplexes can be mapped.

[0020] In sum, a simple force curve records the force on the tip of theprobe as the tip approaches and retracts from a point on the samplesurface. A more complex measurement known as a “force volume,” isdefined by an array of force curves obtained as described above over anentire sample area. Each force curve is measured at a unique X-Yposition on the sample surface, and the curves associated with the arrayof X-Y points are combined into a 3-dimensional array, or volume, offorce data. The force value at a point in the volume is the deflectionof the probe at that position (x, y, z).

[0021] Although this example relates specifically to AFM forcemeasurements that use cantilever deflection as a measure of force, thoseskilled in the art will recognize that there are other physico-chemicalproperties that can be measured using substantially similar probes,instrumentation, and algorithms.

[0022] Although SPMs are particularly useful in making suchmeasurements, there are inherent problems with known systems. Inparticular, typical SPMs use conventional fine motion piezoelectricscanners that translate the tip or sample while generating topographicimages and making force measurements. A piezoelectric scanner is adevice that moves by a microscopic amount when a voltage is appliedacross electrodes placed on the piezoelectric material of the scanner.Overall, the motion generated by such piezoelectric scanners is notentirely predictable, and hence such scanners have significantlimitations.

[0023] A conventional AFM 30 including a piezoelectric scanner 32 isshown in FIG. 4. Scanner 32 is a piezoelectric tube scanner including anX-Y section 34 and a Z section 36. In this arrangement, Z section 36 ofscanner 32 is adapted to support a sample 42.

[0024] To make a force measurement, section 34 of scanner 32 translatessample 42 relative to probe 44 of AFM 30 to a selected position (x,y).As noted previously, to actuate scanner 32, sections 34, 36 includeelectrodes placed thereon (such as 38 and 40 for the X-Y section) thatreceive appropriate voltage differentials from a controller that, whenapplied, produce the desired motion. Next, Z section 36 is actuated totranslate sample 42 toward a tip 46 of probe 44, as described inconnection with the force curve measurement shown in FIGS. 1A-1E and 2.Again, as tip 46 interacts with sample 42, a cantilever 48 of probe 44deflects. This deflection is measured with a deflection detection system50. Detection system 50 includes a laser 51 that directs a light beam“L” towards the back of cantilever 48, which is reflective. The beam “L”reflects from cantilever 48, and the reflected beam “L” contacts a beamsteering mirror 52 which directs the beam “L” towards a sensor 54.Sensor 54, in turn, generates a signal indicative of the cantileverdeflection. Because cantilever deflection is related to force, thedeflection signals can be converted and plotted as a force curve.

[0025] Standard piezoelectric scanners for SPMs usually can translate inthree substantially orthogonal directions, and their size can bemodified to allow scan ranges of typically several nanometers to severalhundred microns in the X-Y plane and typically <10 microns in theZ-axis. Moreover, depending on the particular implementation of the AFM,the scanner is used to either translate the sample under the cantileveror the cantilever over the sample.

[0026] The methods and limitations described above pertaining to currenttypical scanners in SPM are in many cases acceptable in applicationswhere a probe microscope is being used in conventional imaging modes inwhich the XY motion is typically periodic and it is acceptable to use arelative measure of Z movement.

[0027] However force spectroscopy experiments typically demand moreprecise control of relative tip-sample motion, particularly in the Zaxis (the axis substantially perpendicular to the sample surface).

[0028] Typical piezoelectric scanners do not exhibit linear motion,i.e., a given change in the applied drive voltage to the piezo willresult in a different magnitude of motion in different areas if theoperating range. Typical piezoelectric scanners also commonly exhibithysteretic motion, i.e., if a particular voltage ramp is applied to thescanner and then the ramp is re-traced exactly in reverse, one findsthat the scanner follows a different position path on the extend versusthe retract. Piezoelectric scanners also “creep,” which means that theycontinue to extend or retract for a period of time after the applieddrive voltage has stopped changing. Piezoelectric tube scanners alsotypically have low resonant frequencies in the Z-axis. Those skilled inthe art recognize that this represents a serious limitation on the rangeof operating speeds for which the scanner is useful. This is because thepiezoelectric material undergoes complex oscillatory motion when passingthrough and near the resonant frequency.

[0029] Any one or more of these limitations clearly jeopardize theintegrity of the tip-sample motion, and therefore the corresponding datacollected is of marginal usefulness. Overcoming these limitations is oneof the key goals of this invention.

[0030] Alternative means of relative tip-sample motion exist thataddress these concerns, although they can create new problems. Forinstance, sensors can be coupled to piezoelectric scanners by variousmeans well-known in the field. Such sensors can produce a more accuraterecord of motion compared to the more usual assumption that the controlvoltage is representative of the motion. However, adding sensors to ascanner only detects, not corrects, these undesirable motions. However,such sensored scanners can be used in a closed-loop feedbackconfiguration in which the motion is monitored during a change inposition and the applied drive voltage is modified as necessary to makethe actual path of motion more closely match the path specified by thecontrol input signal. Such sensored and closed-loop scanners are mostcommonly implemented in conjunction with a different mechanical designof the scanner known as a piezo-actuated flexure stage (“stage”). Thesestages contain mechanical constraints (flexures) on the motion of thestage intended primarily to constrain the motion of the stage to oneaxis and to mechanically stiffen the stage. This design also presentsmore obvious possibilities for incorporating a sensor than piezoelectrictube designs, although either is feasible in practice. The flexure stageoffers the additional advantage of increasing the resonant frequency ofthe stage relative to a piezoelectric tube scanner with similar range.

[0031] Nevertheless, although the above may seem to suggest a designincluding closed-loop flexure stages in all three axes, in practice sucha design has significant drawbacks. Among the disadvantages of athree-flexure stage design, is that 3-axis flexure stages are muchlarger than a typical piezoelectric tube scanner of similar range due tothe added mass and volume of the constraining mechanism and sensors. Inpractice, larger designs more readily couple outside vibrational andacoustic noise sources into the motion of the scanner, whichsignificantly degrades the scanners usefulness for force spectroscopy.Closed-loop flexure stages are also significantly more expensive thanpiezoelectric tube scanners of similar range.

[0032] Therefore, the use of flexure stages for all three axes is notdesirable for the design of a compact, low-noise, relatively inexpensiveinstrument.

[0033] There are also drawbacks associated with the methods employed tomake conventional force curve measurements. Experimentally, a forcecurve measurement is made by applying, for example, a cyclical trianglewave voltage pattern to the electrodes of the Z-axis scanner as shown inFIG. 5A. The triangle wave drive signal causes the scanner to expand andthen contract in the vertical direction, generating relative motionbetween the probe and the sample. In such a system, the amplitude of thetriangle wave as well as the frequency of the wave can be controlled sothat the researcher can linearly vary the distance and speed that theAFM cantilever tip travels during the force measurement. In FIG. 5B, adrive signal similar to that shown in FIG. 5A is illustrated. However,in this case, the drive signal includes a pause between each change inthe direction of Z scanner motion. In each case, the drive signal iscyclical. However, oftentimes it is desired to modify the parameters ofthe force measurement in a non-cyclical manner, including the speed atwhich the tip-sample separation is modulated, the duration of a pause(to allow molecular binding between tip and molecules on the surface,for example), etc. to analyze forces corresponding to, for example,complex mechanical models of certain samples. In this regard it isnotable that conventional systems often lack flexibility in makingmeasurements that are non-cyclic. Therefore, a system was desired inwhich the flexibility in performing the force measurement is improved.For example, a specific change or rate of change in tip-sample force ora specific value of a tip-sample force may indicate some propertypertaining to the sample in question. In response, it would be desirableto alter a force curve measurement parameter (such as the speed of themovement) in response to a specific measurement condition. Or, forexample it may be desirable to instead of following a path of position(separation) versus time, follow a path of force versus time where theposition (separation) is controlled to produce the desired forceprofile.

[0034] Overall, the field of making force measurements with a probemicroscope was in need of a system including a scanner that is containedin a relatively small package, provides a large Z-axis range withaccurate control of Z motion, and has a relatively high resonancefrequency. Moreover, the field was in need of a system capable ofperforming force measurements according to particular forces measured,and according to particular profiles to maximize the flexibility ofmaking the force measurement. Although not a fundamental requirement forforce spectroscopy, SPM-based system may also be made capable of makingconventional AFM measurements (e.g., topography) by simply switchingmodes of operation.

SUMMARY OF THE INVENTION

[0035] The preferred embodiment overcomes the drawbacks of prior artsystems by providing a force scanning probe microscope (FSPM) thatcombines a flexured Z stage and a piezoelectric tube XY scanner. Moreparticularly, the Z actuator stage is sensored so that proper tip-samplepositioning is maintained, thus maximizing the integrity of the forcecurve measurements. Moreover, the FSPM takes advantage of the ability tomake improved force measurements with more exact positioning by alsobeing adapted to operate according to user-defined position/forceprofiles, as well as having the ability to change a force measurementparameter in response to a trigger condition. In addition to theseautomatic control features, the FSPM also includes a manual controldevice that allows a user to manipulate tip-sample separation accordingto an alert feedback, such as a tactile or audio alert. Although not afundamental requirement for force spectroscopy, the probe microscopebased FSPM may also be made capable of operating in conventional AFMmodes.

[0036] According to a first aspect of the preferred embodiment, apiezoelectric scanner includes a piezoelectric tube that generatesscanner motion in two substantially orthogonal axes defining asubstantially planar surface (“the scan plane”). In addition, thescanner includes a flexured piezoelectric stage that generates scannermotion in a third axis substantially orthogonal to the scan plane.Moreover, the piezoelectric stage is preferably coupled to thepiezoelectric tube.

[0037] According to another aspect of the preferred embodiment, thesensor includes a displacement sensor that detects motion in the thirdaxis and generates a corresponding position signal.

[0038] In another aspect of this embodiment, the scanner is disposed ina probe microscope having a probe and a detection apparatus that sensesmotion of the probe. A sample is positioned such that the scannerproduces relative tip-sample motion, and the displacement sensor ismounted on the stage.

[0039] According to a still further aspect of the preferred embodiment,the stage and sensor are connected to a data acquisition and controlsystem, and wherein the data acquisition and control system generates acontrol signal that drives the stage. Also, the control signal ispreferably generated in response to a user input, where the user inputcan be a position or force profile, which may include triggers.

[0040] According to an alternate aspect of the preferred embodiment, theuser input corresponds to a desired scanner motion, and the controlsignal drives the stage so the scanner motion in the third axis isgenerally the same as the desired scanner motion.

[0041] According to a still further aspect of the preferred embodiment,a force scanning probe microscope (FSPM) includes a piezoelectricscanner having a surface that supports the sample so as to move thesample in three substantially orthogonal directions. The FSPM alsoincludes a displacement sensor that measures movement of the sample in adirection substantially orthogonal to the surface and generates acorresponding position signal so as to provide closed loop positionfeedback. In addition, a probe is fixed relative to the piezoelectricscanner, while a probe motion detection apparatus is employed to sensemotion of the probe. The FSPM also includes a data acquisition andcontrol system that generates a scanner drive signal based on theposition signal and a user-defined input that can change a force curvemeasurement parameter during data acquisition.

[0042] In yet another aspect of the invention, a method of making aforce curve measurement on a sample includes the step of providing anprobe microscope having a probe. Next, the method produces relativemotion between the probe and the sample in response to a user-definedinput defining intended motion in a direction substantially orthogonalto a surface of the sample. In addition, the method includes detectingthe relative motion and comparing the relative motion to thecorresponding intended motion. Further, motion of the probe is measuredwhen the sample interacts with the probe, and a measurement parameter(s)can be changed in response thereto.

[0043] According to another aspect of the invention, a method of makinga force curve measurement on a sample includes the steps of generating adrive signal to modulate a separation between the probe and the sampleaccording to a user-defined input. In addition, the method includesmeasuring the separation and controlling the drive signal in response tothe measuring step. Thereafter, motion of the probe is detected inresponse to the generating step. The method then includes changing ameasurement parameter in response to the detecting step.

[0044] These and other objects, features, and advantages of theinvention will become apparent to those skilled in the art from thefollowing detailed description and the accompanying drawings. It shouldbe understood, however, that the detailed description and specificexamples, while indicating preferred embodiments of the presentinvention, are given by way of illustration and not of limitation. Manychanges and modifications may be made within the scope of the presentinvention without departing from the spirit thereof, and the inventionincludes all such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] A preferred exemplary embodiment of the invention is illustratedin the accompanying drawings in which like reference numerals representlike parts throughout, and in which:

[0046] FIGS. 1A-1E illustrate a probe of an SPM as it is actuated toapproach and retract from a surface so that the tip-sample forces can bemapped;

[0047]FIG. 2 is a plot illustrating force data obtained by the operationshown in FIGS. 1A-1E;

[0048]FIG. 3 is a partially broken away front elevation view of a DNAsample bonded between an AFM tip and a substrate;

[0049]FIG. 4 is a front elevation view of a standard SPM including aconventional piezoelectric tube actuator;

[0050]FIG. 5A is a plot illustrating a cyclical drive signal having aparticular amplitude and speed for making a force curve measurement;

[0051]FIG. 5B is a plot similar to FIG. 5A illustrating a cyclical drivesignal, characterized as including a pause between changes in thedirection of actuation of the Z piezo;

[0052]FIG. 6 is a diagram illustrating a force scanning probe microscope(FSPM) system according to a preferred embodiment of the invention,including a sensored scanner, a control system that maximizes forcecurve measurement flexibility and a mechanical feedback user interface;

[0053]FIG. 7 is a diagram further illustrating the FSPM of the preferredembodiment, including a force mode controller feedback system;

[0054]FIG. 8 is a side elevation view of the FSPM of the preferredembodiment, illustrating the Z sensor;

[0055]FIG. 9 is a cross-sectional front elevation view of the forcespectroscopy scanner shown schematically in FIGS. 6, 7 and 8;

[0056]FIG. 10 is a cross-sectional schematic view of a typical sensoredZ stage preferably implemented in the scanner of FIG. 9;

[0057] FIGS. 11A-11C illustrate alternate embodiments of theconfiguration of the scanner of the FSPM of FIGS. 6-10;

[0058]FIG. 12 is a flow diagram illustrating a method of automaticallyeffecting a force curve measurement with a selected position gradient;

[0059]FIG. 13A is a plot illustrating a user-defined tip-sampleseparation gradient used to drive the Z piezo;

[0060]FIG. 13B is a plot illustrating a force versus time curveassociated with actuating the Z piezo as shown in FIG. 13A;

[0061]FIG. 13C is a force curve generated by combining the position andcantilevered deflection time dependent plots shown in FIGS. 13A and 13B,respectively;

[0062]FIG. 14 is a flow diagram illustrating a method of driving a forcecurve measurement with a selected force gradient;

[0063]FIG. 15A is a plot illustrating a user-defined force gradient usedto control the actuation of the Z piezo;

[0064]FIG. 15B is a plot illustrating a Z piezo position profile thatresults from the force gradient input shown in FIG. 15A;

[0065]FIG. 15C is a force curve generated by combining the timedependent curves shown in FIGS. 15A and 15B;

[0066]FIG. 16 is a flow diagram illustrating a method of automaticallydriving a force curve measurement according to one or more triggerconditions;

[0067]FIG. 17 is a plot illustrating trigger events that cause a changein the drive signal during a force measurement operation, according tothe preferred embodiment;

[0068]FIG. 18A is a plot illustrating an example of a force gradientsuch as that shown in FIG. 15A;

[0069]FIG. 18B is a plot illustrating the Z piezo movement that resultsby inputting the user-defined force gradient shown in FIG. 18A for ahard surface; and

[0070]FIG. 18C is a plot illustrating the Z piezo movement that resultsby inputting the user-defined force gradient shown in FIG. 18A for asoft surface.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0071] Turning initially to FIG. 6, a force scanning probe microscope(FSPM) 100 that provides highly accurate force measurements, includingforce volume measurements, with a high degree of data acquisitionflexibility is illustrated. FSPM 100 includes an atomic force microscope(AFM) 102, a data acquisition and control system 104, and a userinterface 106. Probe microscope 102 includes a probe 108 having acantilever 110 extending from a substrate 112. Cantilever 110 includes afree end to which a tip 114 is coupled so that it extends generallyorthogonal to cantilever 110. Probe 108 is placed in a support (notshown), such as a conventional probe holder, thus collectively defininga probe assembly.

[0072] Next, a sample 116 is mounted on a piezoelectric scanner 118 in aconventional fashion. The sample 116 may, for instance, comprise asingle molecule of a substance of interest. In the preferredimplementation, scanner 118 is used to actuate the sample in threesubstantially orthogonal directions, X, Y, and Z. Notably, movement inthe X,Y directions defines a scan plane generally parallel to themeasured surface of the sample 116, and movement in Z is substantiallyorthogonal to the scan plan. An important aspect of making the forcemeasurements contemplated by the present invention is preciselymodulating the tip-sample separation so the two interact at a particularscan location (x, y). In this regard, some combination of moving boththe tip and the sample could be implemented for providing the relative3-D movement.

[0073] According to the preferred embodiment, and as described infurther detail below, scanner 118 includes an X-Y tube scanner 120 thatis mounted to the AFM chassis (160 shown in FIG. 8) and moves sample 116in the scan plane, i.e., generally parallel to a top surface of scanner118 and the sample surface. As a result, tube 120 operates to position aselected point of the sample beneath, and in-line with, tip 114 of probe108. Scanner 118 also includes a sensored Z actuator 122, which issupported by X-Y tube 120, and shown schematically in FIG. 10.

[0074] During FSPM operation, sensored Z actuator 122 translates sample116 towards (“approach”) and away from (“retract”) tip 114 of probe 108causing interaction between sample 116 and probe 108. In that regard, asprobe tip 114 and sample 116 interact, cantilever 110 deflects. Thisdeflection can be measured as a function of Z movement provided byscanner 118 (for example, one or more approach/retract cycles), and thecorresponding data can be used to plot a force curve.

[0075] The preferred method by which the deflection is measured is byemploying a deflection detection apparatus 123 that directs a light beam“L” generated by a source 124 towards the back of cantilever 110. Thebeam L is then reflected off cantilever 110 and towards a detector 126.The detector 126 can be, for example, a conventional four-cellphotodetector that generates a deflection detection signal based on theposition of the reflected beam. This signal is then transmitted to dataacquisition and control system 104 that communicates with scanner 118 ina closed loop configuration to modulate the tip-sample separation basedon a particular set of imaging parameters, as described in furtherdetail below. Preferably, the light beam is produced by a low noise, lowcoherence length light source 124 (i.e., laser or super luminescentdiode).

[0076] Notably, such a probe microscope may be operated in a mode wheresome other probe motion besides deflection (e.g., oscillatory motion) ismeasured, or wherein the motion (i.e., deflection) of the probe ismaintained at some setpoint by application of some external force (i.e.,laser pressure), and thus the magnitude of this external force isrelated to the tip-sample interaction.

[0077] As described in greater detail below in conjunction with FIGS. 9and 10, X-Y scanner 120 is preferably a piezoelectric tube scanner thatis coupled to sensored Z actuator or stage 122 via an appropriatecoupling. Sensored Z actuator 122, on the other hand, is preferably apiezoelectric flexured stage actuator constructed from a metal mass andincluding flexure points that provide constrained motion in the intendeddirection, i.e., in the vertical or “Z” direction in this case. Thecombination of a tube scanner and a flexure stage actuator providessignificant advantages in making force measurements, including scanrange, predictable, repeatable motion, and high resonance frequency,detailed below.

[0078] With continued reference to FIG. 6, data acquisition and controlsystem 104 communicates with scanner 118 to actuate the scanneraccording to particular force measurement parameters. More particularly,control system includes a force controller 128, an AFM controller 130and a computer 132. Force controller 128 operates to modulate tip-sampleseparation and ensure that the desired Z actuator motion is beingmaintained via feedback from a sensor (162 in FIG. 8) that monitorseither the actual motion of sample 116, or the tip-sample separationdirectly. Force controller 128 is also coupled to SPM controller 130that controls motion of scanner 118 to image surface characteristics,for example, to obtain a topography image using a selected SPM mode ofoperation (e.g., oscillating mode).

[0079] Force controller 128 and SPM controller 130 further communicatewith computer 132 which provides, in at least some embodiments of thepresent invention, instructions to controllers 128, 130 according todesired experiments. In general, desired force measurement parametersare communicated to data acquisition and control system 104, so as toachieve flexibility in making force measurements on a wide range ofsamples (see FIGS. 13A-C, 15A-C, and 17 and their correspondingdescriptions) and acquire a permanent record of the data.

[0080] Note that the exact nature and routing of the signals and thevarious controllers to which are referred are relatively unimportant.For instance, control and data channels could be transmitted as analogor digital signals. The separation of control functions into threenominal controllers/computers could just as well be accomplished with asingle controller/computer in hardware or software. In short, thoseskilled in the art could readily conceive other schemes ofimplementation for the control and data acquisition algorithms describedwhich would still not deviate from the spirit and scope of theunderlying inventive concept.

[0081] In this regard, force measurement parameters may be modifiedaccording to communications from user feedback interface 106 to eithercomputer 132 for automatic control, or directly to force controller 128for manual control. For example, the control provided by control system104 may be defined by a user waveform input 134, or a user trigger input136, according to the operator's desired force measurement, as describedin detail below in conjunction with FIGS. 12-17. On the other hand,rather than providing computer control of desired force measurementparameters, these parameters can be communicated directly with the fourcontroller 128 via a mechanical feedback user interface 140.

[0082] Mechanical feedback user interface 140 of system 100 is coupledto force controller 128 to allow the user to manually adjust theactuation of scanner 118. Therefore, the user can correspondingly adjustthe tip-sample separation. Mechanical feedback interface 140 preferablyincludes a manipulatable device such as a rotary knob 142 which a usermanually manipulates to displace the sample (or probe as appropriate forthe particular implementation) to cause sample 116 to interact with tip114 of the probe 108. In this way, the user can essentially “feel” thesample structure and properties associated with sample 116, based on ameasured force that is fed back to manual interface 140 as an alert. Inthe preferred embodiment, this alert signal can be used to alter acharacteristic associated with manipulating knob 142, e.g., torque. Theoperator can then adjust tip-sample separation to stretch molecules,observe unfolding/refolding of protein domains, etc. Manual interface140 preferably also includes a display 144 so the FSPM operator canmonitor quantitative values associated with the forces being sensed.

[0083] Still referring to FIG. 6, computer 132 is coupled to a display138 to present the actual force, force volume, or SPM image data to theFSPM operator. Moreover, computer 132 of system 104 communicates with akeyboard/mouse or other suitable user interface 146 to allow thesevarious options to be selected by an operator via, for example, agraphical user interface (GUI) (not shown).

[0084] Turning to FIG. 7, FSPM 100 is shown in further detail. Inparticular, the force feedback mechanism of the preferred embodiment isillustrated. Force controller 128 of data acquisition and control system104 includes a Z position drive/feedback loop 148 including aclosed-loop control block 150, preferably, conventional analog circuitryincluding a comparator and a gain stage (not shown), that generates adrive signal that controls piezoelectric flexured Z actuator 122 tomaintain linear motion of Z actuator 122; in other words, motioncorresponding to the desired Z motion as defined by the user input.Control block 150 generates Z-stage drive signal based on two inputs, afirst being the actual Z motion measured by sensored Z actuator 122, andthe second being the desired Z actuator motion transmitted automaticallyfrom computer 132 as a Z position input waveform 154 as detailed below,or manually from manual input device 140.

[0085] With respect to the desired Z actuator motion, flexibility inmaking corresponding force curve measurements is achieved by controllingthe tip-sample separation via Z actuator 122. This motion of Z actuator122 can be defined by a standard input for making force curvemeasurements, such as a cyclic triangle wave, as described previously(FIG. 5A), or it could be a more complex user-defined input. Forexample, a deflection force feedback block 152 may be employed inconjunction with the Z position waveform 154 to maintain a particularforce or force profile, or to change a force measurement parameter inresponse to a “trigger” condition, as shown and described in conjunctionwith FIGS. 14, 15A-C, 16 and 17. Alternatively, the Z position waveformblock 154 can input an appropriate profile to computer 132 that can thenbe communicated to force controller 128 to define a predetermined Zposition profile over time (FIGS. 12 and 13A-C). In yet anotheralternative, rather than automatically controlling Z actuator 122 tomake selected force measurements, the desired actuator motion may becontrolled manually during acquisition of force data by an operator viamanual input device 140.

[0086] Referring to FIGS. 6 and 7, in operation, as tip-sampleseparation is modulated (in the preferred embodiment, by actuating Zactuator 122), probe 108 interacts with sample 116. As a result, themotion of cantilever 110 of probe 108 changes and this change in motionis detected by detector 126. Detector 126, as described previously,generates a probe motion signal (for example, if detector 126 is a splitphotodiode, the quantity (A−B)/(A+B) defines the deflection) that istransmitted to computer 132. Computer 132 includes a detectormeasurement circuit 158 that receives the deflection detection signaland, in response, determines the force acting on probe 108. Deflectionforce feedback block 152 of computer 132 then determines whether aparticular force is being maintained (force profile input), or whether atrigger condition is satisfied. In response, feedback block 152generates and transmits an appropriate control signal to forcecontroller 128 (FIG. 6), which generates a Z-stage drive signal tocontrol actuator 122 translation according to the user's specificationsfor that particular force measurement, i.e., to maintain or change theforce measurement parameters, such as direction of Z motion, speed,etc., as described further below. Computer 132 may also include one ormore Z-position input waveforms that may be selected by an operator(again, preferably via a GUI) to allow the user to flexibly control theactuation of the Z piezo according to a particular experiment to beperformed (FIGS. 12 and 13A-C).

[0087] The position input to control block 150 input is the actualmotion of Z actuator 122, as measured by a sensor (162 in FIG. 8).During a force measurement, the sensor measures the motion of Z actuator122 and generates an associated Z actuator position signal that ittransmits to control block 150 of force controller 128. Control block150 then determines whether the actual translation of Z actuator 122 isthe same as (i.e., linear with) the instructed Z actuated movementprovided by either computer 132 or manual input device 140. If not,control block 150 generates an appropriate Z-stage drive signal tocorrect any Z translation that does not correspond to the user's input.In this way, precision in modulating the tip-sample separation ismaintained.

[0088] In FIG. 8, the sensor feature aspect of scanner 118, and inparticular Z actuator 122, is shown. SPM 102 of system 100 includes achassis 160 to which the deflection detection system 123 and the probeassembly, including probe 108 and mount 117, are coupled. Chassis 160also supports scanner 118, which is fixed thereto. Again, the X-Ytranslation of scanner 118 is provided by piezoelectric tube scanner120, while the Z translation is provided by flexured piezoelectricactuator 122. To monitor the translation of Z actuator 122, a sensor 162is preferably coupled to Z actuator 122. Ideally, sensor 162 is acapacitive sensor that measures the translation of the Z actuator 122(and, hence, translation of the sample 116) by measuring the capacitancechange caused by a change in the separation of the plates of thecapacitor. Referring briefly to FIG. 10, one arrangement of the platesof capacitive displacement sensor 162 is shown. An upper plate 163 isfixed to a stationary portion of flexure stage 122, while a lower plate164 is fixed to a translatable portion 168 of stage 122. As a result,when the Z actuator 122 is actuated, movement of translatable portion168 (and thus sample 116) causes the perpendicular separation betweenplates 163, 164 to be modified. This change in separation is measured todetermine actual Z translation causes a corresponding change in themeasured capacitance. Notably, because the flexure stage from the Zactuator 122 is robust, the separation between plates 163, 164 alongtheir surface areas remains constant (i.e., they maintain their parallelrelationship) when Z actuator 122 is actuated. Moreover, although aparallel plate capacitor is preferred, sensor 162 could be analternative type of sensor such as a piezo-resistive sensor mechanicallycoupled to a reference and the translatable portion of the piezoelectricZ-stage (i.e.—LVDT, or strain gauge sensors).

[0089] As noted above, inaccuracies in tip/sample positioning can arisedue to residual mechanical effects such as hysteresis, creep, thermalexpansion, etc. that can act on all or part of the AFM system. Because,in making force measurements, it is the tip-sample separation that iscritical, an arrangement that eliminates or minimizes these effects wasdesired. In this regard, an alternative to the sensor arrangementdescribed above, also shown in FIG. 8, includes coupling a total systemproximity sensor 165, such as a capacitive sensor, between the sampleand the probe itself to provide a direct measure of tip-sampleseparation. As a result, by measuring the actual tip-sample separationbetween sample 116 and tip 114 with sensor 165, position feedback loop148 (FIG. 7) maintains a high degree of positioning accuracy. Such atotal system proximity sensor 165 essentially eliminates the effects ofhysteresis, creep, and thermal expansion because these effects occurwith respect to the sample and probe assembly simultaneously. If theparallel plates of a capacitive sensor are coupled to the sample andprobe assembly, respectively, these potentially damaging effects canceleach other out when tip-sample separation is measured.

[0090] Next, a detailed view of force spectroscopy scanner 118 is shownin FIG. 9. Scanner 118 provides movement of a sample (116 in FIG. 6, forexample) in three orthogonal directions, which we refer to hereinafteras movement in X, Y and Z. Note that scanner 118 operates to move sample116 underneath probe 108 (FIG. 6), and that movement in X and Y definesa plane generally parallel to a surface of the sample. Again, althoughforce measurements are made at a particular sample location (x,y),movement in the XY plane is required for analyzing different regions ofthe sample. By generating force curves at a variety of XY locations ofthe sample, a force volume image may then be generated, for example.

[0091] Scanner 118 includes a scanner mounting base 169 that is coupledto the chassis (160 in FIG. 8), and that serves as a reference forposition measurements. A scanner core 172 extends upwardly from scannermounting base 169 and is coupled thereto. Scanner core 172 defines atubular structure that encloses and protects piezoelectric XY tubescanner 120, as well as flexured Z actuator 122. Scanner core 172 ispreferably made of a metal, ideally a commercially available steel suchas INVAR.

[0092] Z actuator 122 is coupled to XY tube scanner 120 via an XY-Zcoupling 174. XY-Z coupling 174 is a cap that is positioned over the topof tube scanner 120 and that provides a mounting surface 176 for fixingflexured Z actuator 122 to XY tube scanner 120 conventionally. When theactuator 122 is assembled, axes passing through the center of each ofthe coupled scanner portions are generally collinear.

[0093] In this arrangement, translation of XY tube scanner 120 causescorresponding movement of flexured Z actuator 122. Notably, bypositioning tube scanner 120 beneath Z actuator 122 the X-Y movementproduced by tube scanner 120 is amplified at a free end 178 of scanner118.

[0094] Notably, a conventional piezoelectric tube scanner, such asscanner 120 employed in the preferred embodiment, provides a limitedrange of motion, such that the scan range (i.e., in the XY plane)provided by scanner 118 alone is less than ideal. However, in thepreferred design shown in FIG. 6 (and in more detail in FIG. 9), greaterXY scan range is achieved due to the fact that the object being moved byscanner 118 is displaced from tube scanner 120 a distance generallyequal to the length of sensored Z actuator 122, which is supported bytube scanner 120. In other words, scanner 118 mechanically amplifies theXY range afforded by scanner 118.

[0095] It follows that the larger distance between tube 120 and theobject being moved, in this case a sample placed at the free-end 178 ofscanner 118, the larger the amplification. In this regard, to furtheramplify movement of free end 178 of the scanner 118 produced by the XYtube scanner, a cylindrical Z extension 180 can be included, as shown inFIG. 9. Z extension 180 has opposed ends including a first end 182 whichis fixed to a top portion 186 of Z actuator 122 via a mounting flange orring 188, and a second end 184 that is configured to support a sampleholder 185. As with the X-Y tube scanner 120 and Z actuator stage 122, Zextension has a central axis collinear with the central axis of scanner118. The sample holder is conventional in the art, such as that shown inU.S. Pat. No. Re 34,485. As with piezoelectric Z actuator 122, Zextension 180 is preferably made of INVAR. To minimize the mass ofextension 180, and thus prevent compromising the strength of scanner118, a plurality of holes 190 are formed therein.

[0096] In FIG. 10, a typical flexured Z actuator stage 122 of forcespectroscopy scanner 118, which is adapted for providing Z positionfeedback as described previously, is shown schematically. Z actuator 122includes a mounting surface 192 that is coupled to X-Y tube scanner 120as shown in FIG. 9 with XY-Z coupling 174. Z actuator 122 is preferablya metal block (e.g., Invar) having portions thereof removed to create afixed section or frame 166 and a translatable section 168 coupled toprovide constrained motion in Z, i.e., in a direction perpendicular tosurface 120. Linking fixed and translatable portions 166, 168,respectively, are weakened points that permit movement of the metal masswhen forces are applied thereto. In particular, the metal block includesa series of flexure points 194, 196, 198, 200 formed to constrain motionof translatable portion 168 in a plane orthogonal to the vertical or Zdirection (i.e., in the XY plane), while allowing motion of portion 168in the vertical direction.

[0097] More particularly, each of the flexure points 194, 196, 198, 200comprises a web of metal that can “flex” to allow a sample (116 in FIG.6) mounted on a translating/mounting surface 170 of translatable portion168 to be translated in a direction orthogonal to mounting surface 170.Again, flexure points 194, 196, 198, 200 are coupled to fixed portion166 and support center translatable section 168 of scanner 118.

[0098] Translatable section 168 defines mounting surface 170 and alsodefines a lower contact surface 202 that interfaces with, preferably, apiezo stack 204 mounted within Z actuator intermediate a surface 206 offixed portion 166 and contact surface 202. Piezo stack 204 is aconventional piezoelectric component that produces motion in a selecteddirection in response to appropriate voltages applied to electrodesplaced on the piezoelectric material of piezo stack 204. Piezo stack 204expands and contracts in response to the applied voltage signals suchthat the mechanical motion is transferred to center section 168 of Zactuator 122 via surface 202. Section 168, in turn, moves vertically asflexure points 194, 196, 198, 200 flex. In this case, piezo stack 204 isconfigured to move in a direction substantially orthogonal to mountingsurface 120 in response to the control voltages.

[0099] As noted above, to measure the motion in Z provided by actuator122, first plate 164 of capacitive displacement sensor is fixed totranslatable section 168 of Z actuator 122, while opposed plate 163 ismounted to a surface 165 of fixed portion 166 of Z actuator 122. As aresult, movement of translatable section 168 relative to fixed portion166 can be precisely measured as a change in capacitance due to a changein the perpendicular separation “D” of plates 163, 164 of capacitivedisplacement sensor 161. More particularly, capacitance is proportionalto one over the separation distance, i.e., C=εA/D, where “D” is theperpendicular distance between the parallel plates, thus providing ameasure of Z translation. Notably, plates 162, 164 of sensor 161preferably are rings.

[0100] In operation, a displacement signal is generated by sensor 161and fed back to Z position feedback loop 148 (FIG. 7) to determinewhether the voltage applied to piezo stack 204 resulted in the intendedmotion. If not, one or more a correction Z-stage drive signals can begenerated to provide the intended motion of sample 116. Again, thisaspect of the preferred embodiment allows precise control of the scannerwhich is critical to achieving flexibility in making force measurements,to be described below.

[0101] Notably, flexured Z actuator 122 shown in FIG. 10, is configuredaccording to the schematic shown in FIG. 8. However, as noted above,when a sample coupled to surface 170 is translated in the verticaldirection, effects due to non-linearity, hysteresis, creep, drift, etc.,can contribute to the Z motion provided by actuator 122, thus causingpositioning/spacing problems. Therefore, a sensor arrangement as shownin phantom in FIG. 8 may be preferred for measuring actual Z motionunder certain environmental conditions, conducting particularexperiments, etc.

[0102] Scanner 118 also provides a high resonant frequency,three-dimensional actuator that achieves optimum performance in x,y scanrange and true Z motion in a small package. In the latter regard, theheight of sensored Z actuator 122 is approximately 2″ such that once theactuator 122 is coupled with piezoelectric XY tube scanner 120, scanner118 is maintained in approximately the same package as a conventionalXY-Z tube scanner.

[0103] Overall, scanner 118 produces large Z position range, withsubstantial X-Y range, while being contained in a small package.Moreover, scanner 118 is readily adapted for Z sensing. As a result,scanner 118 is adapted to provide closed loop monitoring of tip-sampleseparation, while minimizing noise problems and having a high resonantfrequency with respect to Z positioning (mechanical flexure driven bypiezo versus a simple piezo stack or tube), which is particularlyimportant when making force measurements.

[0104] Next, alternative configurations of force spectroscopy scanner118 are shown in FIGS. 11A-11C. In FIG. 11A, a scanner 210 includes asectioned Z actuator (tube or stack) 212 that is disposed on top of aflexured Z actuator 214 which, in turn, is mounted on an XY tube 216.Because XY tube 216 is arranged at the bottom of scanner 210, arelatively wide range of XY scanning capability is achieved, for reasonsdescribed previously. Z tube 212 is adapted to accommodate a sample (notshown) and provides motion generally orthogonal to a surface of thesample to modulate tip-sample separation according to the user'srequirements. In this arrangement, flexured Z actuator 214 may providecoarse adjustment of tip-sample separation, while Z tube 212 can beimplemented to provide a fine adjust of the tip-sample separation, i.e.,movement of the sample. This may be particularly desirable when workingwith delicate samples. Or, Z tube could be driven by a high frequencyoscillation to accommodate different modes of operation such as a modesimilar to that described in the literature as “fly-fishing” in whichthe tip is oscillated at a relatively small amplitude and relativelyhigh frequency (via acoustic or magnetic “AC” or TappingMode) while thetip-sample separation is reduced, with the desired result being that theprobe “snags” or “catches” a single molecule on the surface. Theaddition of a second Z axis piezo would allow a similar technique wherethe user can instead use the second piezo to provide the lowamplitude/high frequency oscillation.

[0105] In the alternative shown in FIG. 11B, scanner 220 includes aflexured Z actuator 224 that is positioned intermediate a sectionedpiezoelectric Z tube 226 and a piezoelectric XY tube 222 on which thesample resides. Unlike the previous cases, in this arrangement, the Zactuators 224, 226 move the XY tube actuator 222. Therefore, scanner 220provides the flexibility of the scanner shown in FIG. 11A. However, XYrange of scanner is compromised, as it is disposed nearest the object tobe translated, i.e., the sample. Finally, as shown in FIG. 11C, asimilar arrangement of a flexured Z actuator 236, an XY tube actuator234, and a sectioned Z tube actuator 232 is shown. In this case,flexured Z actuator 236 is fixed to the SPM chassis to provide anactuator having superior strength to the previous embodiments. Moreover,XY scanning range is larger than scanning range of actuator 220 in FIG.11B due to the fact that the perpendicular distance between the XY tubescanner 234 and the sample (disposed on the sectioned Z tube actuator)is larger. In each of the alternative configurations in FIGS. 11A-11C,to best determine tip-sample separation, the preferred Z sensing methodincludes utilizing sensor 165 shown in phantom in FIG. 8, where a directmeasure of separation is made.

[0106] By utilizing the sensors of the preferred embodiment, FSPM 100precisely controls the Z movement of the actuator 118, monitoringwhether the Z movement is in the intended direction, such thatsignificant flexibility in making force curve measurements can beachieved. In particular, according to a further aspect of the preferredembodiment as shown in FIGS. 12-17, methods are disclosed for altering aforce measurement parameter based on a selected user-defined input.

[0107] Turning initially to FIG. 12, a method 250 of making a forcecurve measurement by driving the tip-sample modulation with a tip-sampleseparation gradient includes, initially, a start-up and initializationBlock 252. Next, in Block 254, a signal corresponding to a first pointof a defined input (in this case, a tip-sample separation gradient) istransmitted to the force controller (128 in FIG. 7). Then, method 250generates a drive signal based on the tip-sample separation gradient forthat point in Block 256. This drive signal is then applied, in Block258, to the scanner (118 in FIG. 6).

[0108] Method 250 then measures the Z position of the sample in Block260 (FIG. 8). Next, in Block 262, the method determines whether themovement in Z corresponds to the user-defined input (i.e., closed-loopZ-positioning). If not, a new drive signal to correct the Z motion isgenerated in Block 264 and method 250 is returned to Block 258 to applythe new drive signal to the scanner. If, on the other hand, movementcorresponds to the user-defined input, cantilever deflection is measuredin Block 266. This data is collected and stored in Block 268 and thenplotted as a force versus time curve for that point. Method 250 thenreturns operation to Block 254 to transmit a signal corresponding to theuser-defined input for the next point of the position gradient. Bycombining the position gradient and the force gradient (Block 258) aforce versus separation profile, i.e., a force curve is generated inBlock 270.

[0109] An example of method 250 in operation is shown in FIGS. 13A-13C.The waveform shown in FIG. 13A corresponds to user-defined input ofBlock 254. And, the corresponding forces between the AFM probe tip andthe sample generated as a result of this position profile are measuredand plotted in FIG. 13B (Block 268). Notably, the velocity of thisactuation is defined by the slope of the separation curve. Moreover,note that the zero (“0”) piezo position corresponds to zero tip-sampleseparation, and that negative slope indicates movements upwardly, i.e.,towards the probe as an increasing negative Z-position. A piezo positionat or below “0” indicates no separation as sample engages the AFM tipand continues to move, potentially causing the cantilever to deflectwhile in contact with the sample. In that case, the tip of the probe mayor may not be penetrating the sample, as may be indicated by themeasured forces (FIG. 13B). On the other hand, as the Z-piezo moves thesample downwardly (positive slope in FIG. 13A), tip-sample separation isincreasing. However, as the Z-piezo is withdrawn past the zero position(initial tip-sample contact), the sample may bind to the tip such thatthere is zero actual tip-sample separation for a time as the cantileverdeflects to follow the downward motion of the sample.

[0110] Referring collectively to FIGS. 13A and 13B, as the sampleapproaches the tip of the cantilever starting at time t₁, the tipexperiences a force (FIG. 13B) at about t₂ where the cantilever beginsto deflect upwardly, which is correspondingly sensed by the deflectiondetection system (see 123 in FIG. 6). The flexured Z actuator 122 (FIGS.6-11) is then caused to translate further in the same direction, i.e.,towards the fixed probe, and at the same velocity (constant slope). Thismovement of the flexured Z actuator is halted at a time t₃ where theprobe experiences a positive deflection or force F₁ as shown in FIG.13B. After holding the Z position constant for a period of time t₄ minust₃ (for example, to allow binding of a molecule to the tip), thedirection of movement of the flexured Z actuator is reversed as thesample is pulled away from the tip. In this case, the positivedeflection of the cantilever is reduced as it passes through the zeropiezo position (time t₅) where the tip is essentially resting on thesample as the Z actuator pulls the sample further away from the probe.As this actuation continues, binding between the tip of the probe andthe sample produces a negative deflection and a corresponding negativeforce that increases to a value F₂ (FIG. 13B), as Z translationcontinues until a time t₆. Notably, if there was no binding of thesample to the tip, the actual separation between the sample and the tipwould be non-zero.

[0111] At time t₆, this negative deflection of the probe decreases asthe direction of translation of Z motion is reversed, such that thedeflection of the cantilever of the probe again approaches zero. In thiscase, in the case of a titin molecule, the molecule is allowed to“refold,” as Z actuator again moves in a direction towards themicroscope tip. The corresponding measured forces are indicative ofsample properties. This approach/withdraw cycle is repeated until thepiezo is translated away from the probe at a constant velocity at timet₁₁. As a result, the Z actuator moves the piezo until the tip releasesfrom the sample, at which time t₁₂ the force plotted in FIG. 13B returnsto zero, i.e., as the cantilever of the probe returns to its free-airdeflection.

[0112] Thereafter, the position gradient shown in FIG. 13A and thecorresponding force data measured as a function of time (FIG. 13B),which again is measured in response to the selected tip-sampleseparation gradient shown in FIG. 13A, are combined in conventionalfashion to produce the force versus separation curve shown in FIG. 13C.In sum, by controlling the Z actuator position relative to the fixedprobe, different properties of the sample supported by the flexuredactuator can be observed and recorded according to the user'srequirements. For example, in one experiment, forces measured during thestretching and refolding of particular molecules, such as titinmolecules, can be analyzed according to particular models of theirmechanical behavior.

[0113]FIG. 13C illustrates a force curve similar to that shown in FIG.2. Note that the bi-directional arrows on the force curve indicateapproach (decreasing “piezo Z”, i.e., decreasing tip-sample separation)and retract (increasing “piezo Z”). As the sample approaches thecantilever tip, zero force is experienced by the tip such that nodeflection of the cantilever is detected. As the tip begins to interactwith the sample at position z₁ (generally corresponding to time t₂ inFIGS. 13A and 13B) the cantilever begins to deflect upwardly. Thisdeflection is plotted as a positive force. As the sample is translatedfurther towards the tip, the cantilever of the probe deflects further,thus increasing the force detected. As the tip continues to interactwith the sample, the actuator position reaches a point zhd 2 where thedirection of the movement is changed. In particular, the sample ispulled away from the tip, thus causing the measured force to decreaseuntil it reaches a point z₁ where the probe experiences zero force onceagain.

[0114] As the sample is withdrawn further from this zero force position,because in this case the tip binds to the sample, the sample begins to“pull” the probe downwardly as the force continues to increase in theopposite direction. Thereafter, the tip releases from the sample at z₃,such that the probe deflection returns to its free-air zero value.

[0115] Another user-defined profile is illustrated in FIG. 14 as amethod 300 for controlling one or more force measurement parametersaccording to a user-defined force gradient. The method 300 is alsoillustrated graphically with an example in FIGS. 15A, 15B and 15C. Aftera start-up and initialization Block 302, method 300 transmits auser-defined force gradient signal for a particular point of thegradient to the force controller (128 in FIG. 7) in Block 304. Next, inBlock 306, the force controller generates a drive signal based on theuser-defined force gradient for that point (e.g., selecting a velocity,direction and duration of Z actuation), and then applies the drivesignal to the Z actuator in Block 308. As the drive signal is applied,the Z position of the scanner is measured in Block 310 (closed-loopZ-positioning), and method 300 determines whether the movementcorresponds to that dictated by the drive signal in Block 312. If not, adrive signal is generated in Block 314 to correct the Z motion. If so,the deflection of the cantilever is measured, collected and stored todetermine the force on the cantilever in Block 316.

[0116] Method 300 next plots position on a position versus time plot forthat point in Block 318. In Block 320, method 300 determines whether theforce on the cantilever corresponds to the user-defined force input(FIG. 15A, for example, described below) for the particular pointdefined in Block 304. If the force correspondence requirement is notmet, the process is returned to Block 306 to generate a new Z-stagedrive signal based on the user-defined input and the measured deflection(instruction generated and communicated from deflection force feedbackblock 150 in FIG. 7). For example, if the measured force is less thanthe desired force, a signal is transmitted to the force controller toinstruct the force controller to transmit an appropriate signal to the Zactuator to move the sample faster so that the correct cantileverdeflection (i.e., force) for that point is achieved. If, on the otherhand, the force is met, method 300 asks whether each point in the forcegradient has been considered in Block 322. If not, the process returnsto Block 304 to transmit a force control signal for another point of theforce gradient. If data has been obtained for each point in the forcegradient, the collected and stored data (including the force gradientand position plot) are combined and plotted as a force versus a positionprofile of the sample (i.e., the force curve) in Block 324.

[0117] An illustrative example of method 300 in operation is shown inFIGS. 15A-15C, defining another type of user-defined waveform input tocontrol the acquisition of a force curve. Again, method 300 is directedto inputting a force gradient (FIG. 15A) (i.e., a rate of change offorce) and measuring the corresponding position of the piezo required toachieve that force gradient (FIG. 15B). More particularly, withreference to FIG. 15A, between time t₀ and time t₁, the force is heldconstant at a zero value which typically will correspond to bringing thetip and sample into contact, as shown in FIG. 15B for this time range.At time t₁ the force gradient in FIG. 15A instructs the Z piezo actuatorto move so that the force increases linearly from time t₁ to time t₂.Notably, as shown in FIG. 15B, at t₁ the cantilever deflection is zeroand the piezo position is at zero tip-sample separation. Then, thecantilever deflects upwardly during the time t₂ minus t₁ as the actuatormoves further towards the probe with the tip in contact with the sample.Between t₂ and t₃, the force is held constant, and thus the forcecontroller does not cause the Z actuator to move.

[0118] At t₃, the method 300 instructs the Z actuator to move the sampleso that the force is reduced linearly. At the time t₄, the force is zerowhile force controller continues to cause the Z actuator to pull thesample away from the microscope tip. Then, between t₄ and t₅, a linearforce gradient in the same direction provides the instructions to theforce controller for actuating the flexured piezoelectric Z actuator. Itis notable that the actuation of the Z actuator required to maintain thelinear force gradient shown between t₄ and t₅ in FIG. 15A, is nonlinearas shown in FIG. 15B. In other words, force is not directly proportionalto tip-sample separation. As a result, including such a force gradientcan provide useful information that cannot be obtained by using thetip-sample separation gradient shown in FIGS. 13A-13C. Finally, as thelinear force gradient is continuously applied, the Z-stage drive to theZ piezoelectric actuator continues to pull the sample away from the tipuntil point t₅ at which time the tip releases from the sample surfaceand the force on the probe returns to zero. As in FIGS. 13A-13C, thetime dependent curve shown in FIGS. 15A and 15B can be combined toproduce the force versus separation curve shown in FIG. 15C. A notableregion of the force curve of FIG. 15C is between the times t₄ and t₅(labeled (t₄, t₅)) illustrating the binding of the tip to the sample asthe Z-piezo is moved away from the probe. Although the force versusseparation curves shown in FIGS. 13C and 15C are similar, theinformation provided in the measured curves in FIGS. 13B and 15B caneach provide unique, valuable information regarding the particularexperiment being conducted.

[0119] Next, trigger operation of the force SPM is very similar to theuser-defined force gradient shown in FIGS. 13A-13C. However, rather thanbeing predetermined as is the case with the user-defined force gradient,trigger-based operation can alter the force gradient, real-time, inresponse to a particular condition.

[0120] Referring initially to FIG. 16, a method 400 of trigger operationincludes a start-up and initialization Block 402. Next, the Z-stagedrive signal is generated according to at least one force measurementparameter for a particular point (e.g., a particular point in time onthe trigger profile such as that shown in FIG. 17), in Block 404. Next,the Z-stage drive signal is applied to Z actuator to move the actuatorfor a particular amount of time, and in a selected direction and speedin Block 406. Next, in Block 408, method 400 measures the Z position ofthe sample to determine tip-sample separation. As part of theclosed-loop positioning, in Block 410, method 400 determines whether theZ position corresponds to the drive signal. If it does not, method 400generates a new Z-stage drive signal to correct Z motion so itcorresponds to the intended motion defined by Block 404. If, on theother hand, Z position corresponds to the drive signal, the cantileverdeflection for that point is measured and stored in Block 414. Next, inBlock 416, method 400 collects and stores the corresponding positiondata.

[0121] Continuing, method 400 next determines whether a triggercondition has been met. If not, the process returns to Block 404 togenerate another Z-stage drive signal for another particular point,i.e., the next point in time. If, on the other hand, the triggercondition is met, method 400 changes at least one force measurementparameter of the force curve acquisition process in Block 420. Once alltrigger conditions of the trigger profile (for example, FIG. 17) havebeen met, method 400 is terminated and a position versus time plot iscombined with the trigger profile to generate a force curve.

[0122] Turning to FIG. 17, a force versus time curve generated accordingto the method 400 illustrated in FIG. 16 is shown. More particularly, asthe computer instructs force controller to generate a Z-stage drivesignal, the sample approaches the tip of the probe of the AFM and zeroforce on the probe is measured by the deflection detection system.Thereafter, as the tip begins to interact with the sample (e.g., amolecule) at time t₁, the force increases linearly to a value F₁(positive deflection force) as the force spectroscopy actuator narrowsthe tip-sample separation.

[0123] At time t₂, a first trigger condition (a predetermined force) ismet at a positive deflection force equal to F₁. In response, tip-sampleseparation is kept at the t₂ value for a time period t₃ minus t₂ (forexample, to allow time for the tip to bind to the sample). At t₃, basedon the first trigger, the computer transmits a signal to the forcespectroscopy scanner to begin withdrawing the sample from the tip toincrease tip-sample separation. At t₄, the tip of probe of FSPM isresting on the sample surface such that there is no deflection of thecantilever of the probe measured. As the separation is increasedfurther, a linear force gradient is measured during a time t₅ minus t₃as the tip passes through the zero force point at t₄. At that point,upon detection of the negative deflection force at t₅, Z movement of theforce spectroscopy scanner is halted for a period of time equal to t₆minus t₅. At t₆, computer instructs force controller to continue to“pull” on the sample based on the second trigger at a particularvelocity to produce the force gradient shown in region “A”. Tip-samplebinding deflects the cantilever downwardly, and these forces aremeasured between times t₆ and t₇, producing as described previouslyuntil the tip separates from the sample at t₇ and the force between thetip and sample is again returned to zero as the tip is no longerinteracting with the sample.

[0124] A straightforward example of using a force gradient to determinea sample property is illustrated in FIGS. 18A-18C. FIG. 18A defines aforce gradient that is used to control the Z actuator similar to thatshown in FIG. 15A. For a hard surface, illustrated with the Z positionprofile in FIG. 15B, the Z actuator is caused to move in a generallylinear fashion both on the approach 450 (zero then increasing force) andretract 452 of the tip-sample separation. More particularly, to followthe force profile shown in FIG. 18A, the actuator moves linearly until atime t₁ defining a zero position where the tip begins to contact thesample, at which the time the force on the tip begins to increase. Inthe region from t₁ to t₂ the force gradient increases linearly (FIG.15A) and the actuation of the Z actuator moves linearly as well (FIG.15B) as the tip presses against the hard surface. At the peak force F₁(at time t₂) the actuation of the flexured Z actuator is at a peak Z₁such that the cantilever is deflected upwardly at its maximum. At thispoint, the instruction based on the force gradient is to actuate Ztranslation to reduce the force to a zero level. The motion of the Zactuator, in response, is linear to the zero point (time t₃) where thetip barely contacts the sample surface. As the sample is furtherwithdrawn from the tip, the force remains at zero as there is no bindingbetween the sample and tip.

[0125] In each of the methods illustrated in FIGS. 12, 14 and 16, it isnotable that the precise Z-positioning provided by scanner 118 of thepreferred embodiment enables the use of a wide range of user-definedinput profiles, thus allowing FSPM 100 to target measuring particularmechanical properties (e.g., based on sample models) of a wide range ofsamples.

[0126] In contrast, for a soft surface, as shown in FIG. 18C, to achievethe same peak force F₁ on the tip of the probe, the force profile shownin FIG. 18A causes the Z piezo to move to a position Z₂ in a non-linearpath, whereby the value of Z₂ is much greater than Z₁, thus indicating asofter sample. As a result, with the same force profile, two types ofsamples can be investigated by considering their position profiles.

[0127] Although the best mode contemplated by the inventors of carryingout the present invention is disclosed above, practice of the presentinvention is not limited thereto. It will be manifest that variousadditions, modifications and rearrangements of the features of thepresent invention may be made without deviating from the spirit andscope of the underlying inventive concept.

What is claimed is:
 1. A piezoelectric scanner comprising: apiezoelectric tube that generates scanner motion in two substantiallyorthogonal axes defining a substantially planar scan plane; a flexuredpiezoelectric stage that generates scanner motion in a third axissubstantially orthogonal to said scan plane; and wherein saidpiezoelectric stage is coupled to said piezoelectric tube.
 2. Thescanner of claim 1, further comprising: a displacement sensor thatdetects motion in the third axis and generates a corresponding positionsignal.
 3. The scanner of claim 2, wherein said position signal isindicative of a tip-sample separation.
 4. The scanner of claim 2,wherein said displacement sensor is a capacitive sensor.
 5. The scannerof claim 2, wherein said displacement sensor is coupled between a probeof a probe microscope and a sample.
 6. The scanner of claim 5, whereinone of the sample and the probe is coupled to the scanner.
 7. Thescanner of claim 2, wherein the scanner is disposed in a probemicroscope having a probe and a probe motion detection apparatus thatsenses motion of the probe, and wherein a sample is moved relative tothe probe by the scanner, and wherein said displacement sensor ismounted on said stage.
 8. The scanner of claim 7, further comprising: acontroller; and wherein said controller generates a control signal thatdrives said stage, and wherein the control signal is generated inresponse to a user-defined input.
 9. The scanner of claim 8, whereinsaid stage generates scanner motion in the third axis in response to theposition input signal.
 10. The scanner of claim 8, wherein the controlsignal drives said stage so the scanner motion in the third axissubstantially matches that defined by the user-defined input.
 11. Thescanner of claim 8, wherein the user-defined input corresponds topredetermined scanner motion, and wherein said control signal drivessaid stage so the scanner motion in the third axis is generally the sameas the predetermined scanner motion.
 12. The scanner of claim 8, whereinthe user-defined input is a path defined by position versus time. 13.The scanner of claim 8, wherein the user-defined input is a path definedby force versus time.
 14. The scanner of claim 8, wherein theuser-defined input includes a trigger condition.
 15. The scanner ofclaim 14, wherein the trigger condition is one of a predetermined force,a predetermined change in force, and a predetermined force gradient. 16.The scanner of claim 15, wherein, during a force curve measurementoperation defined by a measurement parameter, the probe motion detectionapparatus senses motion of the probe corresponding to interactionbetween the probe and the sample, and wherein the trigger condition ismet by the probe motion so as to change the measurement parameter. 17.The scanner of claim 2, further comprising a closed-loop feedbackcontroller that generates a drive signal in response to the positionsignal to produce scanner motion in the third direction corresponding toa user-defined input.
 18. The scanner of claim 1, wherein said stage ispositioned so as to mechanically amplify the motion of the scanner inthe first plane.
 19. The scanner of claim 1, wherein one of saidpiezoelectric tube and said stage is configured to be coupled to asample.
 20. The scanner of claim 1, wherein said stage is configured tobe coupled to a sample, and said piezoelectric tube moves the sample inthe scan plane.
 21. The scanner of claim 1, further including apiezoelectric Z actuator that generates scanner that generates scannermotion in the third axis in addition to that generated by thepiezoelectric stage.
 22. The scanner of claim 21, wherein saidpiezoelectric tube is disposed intermediate said Z actuator and saidstage.
 23. The scanner of claim 21, wherein said piezoelectric tube issupported by said Z actuator and said stage.
 24. The scanner of claim 1,wherein the scanner provides relative motion between a probe of a probemicroscope and a sample.
 25. The scanner of claim 24, wherein the probeis coupled to the scanner and the sample is fixed relative to the probe.26. A probe microscope including a probe that interacts with a samplehaving a surface, the microscope comprising: a piezoelectric scannerproducing relative motion between the sample and the probe in threesubstantially orthogonal directions; a displacement sensor that measuresthe relative motion in a direction orthogonal to the surface andgenerates a corresponding position signal; a probe fixed relative tosaid piezoelectric scanner; a probe motion detection apparatus thatsenses movement of the probe; and a controller that generates a scannerdrive signal based on said position signal.
 27. The microscope of claim26, wherein said displacement sensor is mounted on said scanner.
 28. Themicroscope of claim 26, wherein said displacement sensor is coupledbetween said probe and said sample.
 29. The microscope of claim 26,wherein the scanner drive signal is generated in response to apredetermined user-defined input that defines scanner motion.
 30. Themicroscope of claim 29, wherein the user-defined input is a path definedby position versus time.
 31. The microscope of claim 29, wherein theuser-defined input includes a trigger condition.
 32. The microscope ofclaim 31, wherein the trigger condition is one of a predetermined force,a change in force, and a rate of change of force.
 33. The microscope ofclaim 26, wherein the scanner drive signal is generated in response to amanual user-defined input.
 34. The microscope of claim 26, wherein thescanner drive signal is generated in response to a predetermineduser-defined input that is a path defined by force versus time.
 35. Amethod of making a force curve measurement on a sample, the methodcomprising: providing a probe microscope having a probe; producingrelative motion between the probe and the sample in response to auser-defined input defining intended motion in a direction generallyorthogonal to a surface of the sample; detecting the relative motion andcomparing the relative motion to the corresponding intended motion;measuring a force on the probe when the sample interacts with the probe;and changing a force curve measurement parameter in response to saidmeasuring step.
 36. The method of claim 35, further comprising the stepof generating relative motion between the probe and the sample inresponse to said detecting step.
 37. The method of claim 35, whereinsaid measuring step includes detecting a motion of the probe.
 38. Themethod of claim 35, wherein said changing step is performed based on theuser-defined input.
 39. The method of claim 35, wherein the force curvemeasurement parameter is a direction associated with said producingstep.
 40. The method of claim 35, wherein the force measurementparameter is a speed associated with said producing step.
 41. The methodof claim 35, wherein the force curve measurement parameter is pausingsaid producing step for a predetermined amount of time.
 42. The methodof claim 38, wherein the user-defined input is a path defined by forceversus time.
 43. The method of claim 42, further including the step ofdetermining a force gradient based on said measuring step performed forat least two points in time.
 44. The method of claim 35, wherein saidproducing step is interrupted prior to said changing step.
 45. Themethod of claim 35, wherein said measuring step is performed for atleast two points in time, and said changing step is performed inresponse to a predetermined change in the force.
 46. The method of claim35, wherein said changing step is predetermined.
 47. The method of claim46, wherein said changing step includes modulating a separation betweenthe sample and the probe so that the measured forces corresponding to aplurality of points in time correspond to a predetermined force profileas a function of time.
 48. The method of claim 47, wherein saidmodulating step is performed using a force feedback loop.
 49. The methodof claim 35, further comprising the step of measuring a separationbetween the sample and the probe as a function of time.
 50. The methodof claim 35, wherein said changing step includes controlling theseparation between the probe and the sample according to a user-definedpath defined by position versus time.
 51. The method of claim 35,wherein said producing step is performed by a sensored Z stage.
 52. Themethod of claim 51, wherein said sensored Z stage is coupled to apiezoelectric tube scanner, said tube scanner providing scanning motionin a plane substantially orthogonal to the third axis.
 53. A method ofmaking a force curve measurement on a sample, the method comprising:generating a drive signal to modulate a separation between a probe andthe sample according to a user-defined input; measuring the separation;controlling the drive signal in response to said measuring step;detecting a force on the probe in response to said generating step; andchanging a force measurement parameter in response to said detectingstep.
 54. The method of claim 53, wherein the user-defined input is anautomatic input.